Reforestation, the controlled regeneration of forests, has become an integral part of forest management in order to secure a renewable and sustainable source of raw material for production of paper and other wood-related products. Forest trees can be regenerated by either sexual or asexual propagation. Sexual reproduction of seedlings for reforestation has traditionally been the most important means of propagation, especially with coniferous species. However, inherent biological and economic limitations on the use of sexual reproduction methods for large-scale seed production have caused considerable interest to develop in utilizing asexual methods to propagate conifers; especially economically important conifers of the genera Picea, Pinus, and Pseudotsuga.
One may avoid these biological limitations by using asexual propagation to exercise a very high selection intensity--that is, to propagate only progeny showing a very high genetic gain potential. As desirable progeny have genetic combinations which result in superior growth and performance characteristics, such progeny may be clonally propagated to develop a collection of genetically identical individuals for planting. Clone deployment has the capacity to increase fiber production and assure uniformity in field planting stocks.
The primary asexual propagation methods for trees are vegetative propagation (via either rooted stem cuttings or somatic embryogenesis) and grafting. Although grafting is widely used to propagate selected individuals in limited quantities for seed orchard establishment, it is not applicable to large-scale production for reforestation. Vegetative propagation via rooted stem cuttings or via somatic embryogenesis currently holds the most potential for reforestation of coniferous trees. Both methods contain benefits for forestry operations, depending upon the desired application.
Propagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from small pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Vegetative propagation via somatic embryogenesis has the capability to capture all genetic gain of highly desirable genotypes. Furthermore, these methods are readily amenable to automation and mechanization. Thus somatic embryogenesis has the potential to produce large numbers of individual clones for reforestation.
It was not until 1985 that somatic embryogenesis was discovered in conifers (Hakman et al. 1985) and the first viable plantlets were regenerated from conifer somatic embryos (Hakman and von Arnold 1985). Since 1985, conifer tissue culture workers throughout the world have pursued the development of somatic embryogenesis systems capable of regenerating plants. The goal of much of this work is to develop conifer somatic embryogenesis as an efficient propagation system for producing clonal planting stock en masse. In addition, the embryogenic system interfaces very well with genetic engineering techniques for production of transgenic clonal planting stock of conifers.
The two most economically important conifer genera are Picea (spruce) and Pinus (pine). There are about 30 species of Picea, largely restricted to cooler regions of the northern hemisphere, of which seven species are native to North America. Pinus is the largest and most important genus of conifers, having approximately 95 species scattered over the northern hemisphere. Of these 95 species, 36 are native to North America (Preston 1989).
Those working in conifer somatic embryogenesis have found that there is a striking difference between Picea conifers and Pinus conifers as to the ease with which somatic embryogenesis can be induced and plants regenerated (Tautorus et al. 1991). Indeed, if one evaluates the success of somatic embryogenesis in conifers among species of these two important genera, it is clear that much more success has been achieved with Picea than with Pinus. It is also striking how consistent the success on developing somatic embryogenic systems has been among several Picea species, whereas the recalcitrance of Pinus has been equally consistent across several species.
Among Picea species embryogenic culture initiation frequencies are relatively high; as high as 95% from immature zygotic embryos and as high as 55% from mature zygotic embryos harvested from fully developed, dry seeds (Tautorus et al. 1991). There are numerous reports of production of fully developed somatic embryos among Picea species, and several reports of establishment and growth of Picea somatic embryo plants in soil. Researchers at the British Columbia Research Corporation have reported on establishment of interior spruce (a mixture of Picea glauca and Picea englemannii) somatic embryo plants under nursery conditions. For example, Webster et al. (1990) reported over 80% survival and establishment in nursery conditions of interior spruce somatic embryo plants for most of 71 genotypes tested. Grossnickle et al. (1992) reported the establishment of 40% of 2000 interior spruce somatic embryo plants in nursery conditions. The somatic embryo plants were derived from 15 different genotypes. Researchers at the Weyerhaeuser Company have reported similar success with Norway spruce (Picea abies); over 3000 somatic embryo plants from 17 genotypes have been established in the field (Gupta et al. 1992). Similar success was also reported with Douglas-fir (Pseudotsuga menziesii); over 2000 somatic embryo plants from 6 genotypes of have been established in soil in greenhouse conditions. Thus, conifer somatic embryogenesis workers utilizing Picea species (and commercially important Douglas-fir) have been successful in developing culture initiation, maintenance, and regeneration systems that enable relatively routine production of plants capable of transfer to field conditions. The rapid successes in Picea somatic embryogenesis had led to considerable optimism among researchers that commercial utilization of conifer somatic embryogenesis for production of clonal planting stock of Pinus conifers would be readily achievable.
However, the progress achieved with somatic embryogenesis in Pinus species to date has been much less encouraging than that achieved with Picea species. First and foremost in difficulty is the recalcitrance of Pinus species for initiation of embryogenic cultures. For example, initiation frequencies of about 1 to 5% are routinely cited by those working with Pinus species (Gupta and Durzan 1987a, Becwar et al. 1988, Jain et al. 1989, Becwar et al. 1990). The single report claiming a 54% initiation rate from immature zygotic embryos of Pinus strobus (Finer et al. 1989) has yet to be repeated or duplicated by others working with this species (Michler et al. 1991). Secondly, it is extremely difficult to obtain reliable development of Pinus somatic embryos to the fully developed (cotyledonary) stage. In addition, subsequent production of plantlets has been extremely limited in Pinus species. Tautorus et al. (1991) cited only 3 of 7 reports which indicated plantlets were obtained via somatic embryogenesis in Pinus species. (In contrast, 30 of 43 reports with Picea species reported obtaining plantlets via somatic embryogenesis.) Unlike the reports with Picea species where several systems have shown potential for plantlet production on relatively large scales, the reports of plantlet production from Pinus species have yielded few plants. To our knowledge there is only one report of successful establishment of Pinus somatic embryos in soil (Gupta and Durzan 1987a). The authors of this report have had limited success in obtaining Pinus taeda somatic embryo plants . . . indeed, only one culture genotype was taken to the plantlet stage and only one plant was transferred to soil (see Pullman and Gupta 1991). To date the only published report of higher numbers of germination of Pinus somatic embryos is for Pinus caribaea, where 34 of 69 (49%) germinated (Laine and David 1990). However, the authors did not report establishment of these plants in field conditions.
The recalcitrance seen in the initiation, development, and field establishment of somatic embryo plants in Pinus when compared to other conifers is also true for the establishment of Pinus embryogenic liquid suspension cultures. Shake flask suspension cultures have been established from embryogenic tissues in Picea abies (Hakman et al. 1985), Picea glauca (Hakman and Fowke 1987; Attree et al. 1989), Pseudostuga manziesii (Durzan and Gupta 1987), Picea mariana (Tautorus et al. 1990), and Picea glauca-engelmannii (Tautorus et al. 1992). In these species both the development of stage 3 embryos and recovery of complete plantlets have been accomplished (Hakman and von Arnold 1988; Attree et al. 1990), (Durzan and Gupta 1987; Tautorus et al. 1992). As has been the case with other tissue culture systems in conifers, both Picea and Pseudostuga have historically been much more amenable to the somatic embryogenesis process and, therefore, the establishment of embryogenic suspension cultures in Picea and Pseudostuga are generally considered routine.
However, the genus Pinus has been a much more recalcitrant species in both semi-solid and liquid tissue culture systems. Indeed, there are very few examples of the successful establishment of embryogenic liquid suspension cultures in Pinus--and in only two cases were stage 3 embryos developed from embryogenic liquid suspension cultures. To date, embryogenic suspensions have been reported in Pinus taeda (Gupta and Durzan 1987a and 1987b; Gupta and Pullman 1991; Pullman and Gupta 1991), Pinus strobus (Finer et al. 1989) and Pinus caribaea (Laine and David 1990; Laine et al. 1992).
Moreover, of these reports only in Pinus caribaea (Laine et al. 1992) have suspension cultures given rise to stage 3 embryos that were then germinated to plantlets and subsequently transferred to soil. These plants were obtained from suspension cultures that were either cryostored or non-cryostored and, apparently, these cultures were established from only one or two genotypes. There were no data given as to the actual numbers of stage 3 embryos produced. In contrast, the present method produced large numbers of stage 3 embryos from several different genetic lines. A major difference between this reported process and the present method is that the present method teaches the addition of activated carbon to suspension cultures, while the reported process does not.
In Pinus taeda, Gupta and Durzan (1987a and 1987b) report the development of somatic embryos from suspension cultures--however, apparently these were only globular or torpedo-shaped embryos (therefore presumably stage 1 and stage 2 embryos). No data were presented as to the numbers, if any, of late stage 3 embryos produced. Furthermore, the plantlets that were produced in this report (Gupta and Durzan 1987a) were from embryogenic tissues grown on a semi-solid medium, not from liquid cultures. Finally, in contrast to the present method the reported processes did not utilize activated carbon in suspension cultures.
In a recent patent Gupta and Pullman (1991) report on the development of embryos of Pinus taeda from a suspension culture using activated charcoal and abscisic acid (ABA) on a semi-solid development medium. These were apparently well developed stage 3 embryos, but were from two genotypes only (denoted as genotype A and genotype B). However, the reported process did not utilize activated carbon in its liquid suspension cultures.
Stage 3 embryos were produced In Pinus strobus, but no details were given as to either their morphology or their germination potential (Finer et al. 1989). Also, no data was given on the number of lines tried or on the efficiency of establishment and maintenance of the liquid culture system. This process also did not utilize activated carbon in the suspension culture medium.
Therefore, with the possible exception of Pinus caribaea no stage 3 somatic embryos capable of germination have been successfully produced in the genus Pinus from embryogenic liquid suspension cultures of a range of genotypes or cell lines. Moreover, while stage 3 embryos were produced in Pinus caribaea, the efficiency was extremely low (apparently only 2 plants were produced from 1 or 2 lines) and both plants showed phenotypic abnormalities (Laine et al. 1992).
Prior to the discovery of the present method it has been extremely difficult to establish viable suspension cultures from embryogenic tissue cultures of loblolly pine. For example, a series of experiments have shown that most embryogenic tissue cultures maintained on a semi-solid medium [containing DCR basal salts (Gupta and Durzan 1985 and Table I below) and vitamins with 3.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l N.sup.6 -benzyladenine (BAP) and 2.0 g/l GELRITE.RTM. (gellan gum manufactured by Merck, Inc.)] would not grow when placed in a liquid medium of the same composition minus the gelling agent. Most cultures would not grow or would grow for a short time and die. In fact, out of 92 individual trials only 9 were successful and only 5 out of 18 different genetic lines were successfully maintained in a liquid environment.
Commonly assigned (and allowed) U.S. patent application Ser. No. 08/138,994, filed Oct. 21, 1993, now U.S. Pat. No. 5,413,930, which is hereby incorporated by reference, teaches and claims a process for regeneration of coniferous plants by somatic embryogenesis. The present method improves upon this process by allowing the practitioner to utilize liquid suspension cultures as embryogenic tissue maintenance media.
Therefore, an object of the present invention is to provide an improved method for establishing and maintaining embryogenic liquid suspension cultures for use in somatic embryogenesis processes for plants of the genus Pinus and Pinus interspecies hybrids.
Another object of the present invention is to provide an improved method for the regeneration of coniferous plants by somatic embryogenesis via the utilization of embryogenic liquid suspension cultures.
A further object of the present invention is to provide an improved method for the establishment and maintenance of embryogenic suspension cultures from plants of the genus Pinus and Pinus interspecies hybrids so that these cultures can be further induced to regenerate stage 3 embryos when placed in the development stage, and further germinated and converted to yield viable plants for field planting.